权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Novel Signal Transduction in the Regulation of Vascular Tone and Their Involvement in Diseases

血管张力调节中的新信号转导及其与疾病的关系

基本信息

批准号：
11694261
负责人：
ITO Masaaki
金额：
$ 4.99万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694261/
关键词：
Smooth Muscle Myosin Phosphatase Rho-kinase RhoA CPI-17 MYPT1 平滑節

项目摘要

1. Purified Rho-kinase from smooth muscle, identified as ROKα isoform, could phosphorylate both the regulatory light chain (Ser19) of smooth muscle myosin and MYPT1 subunit of myosin phosphatase (MP). Rho-kinase could be activated by arachidonic acid independently with respect to RhoA. Phosphorylation of MYPT1 at Thr695 induced the inhibition of MP activity. Using a site- and phosphorylation-specific antibody for Thr695, increased levels of MYPT1 phosphorylation at Thr695 by Rho-kinase were observed in cultured vascular smooth muscle cells by agonist stimulation.2. CPI-17, an inhibitory phosphoprotein for MP, could be phosphorylated by two RhoA downstream kinases, namely Rho-kinase and PKN. These phosphorylation converted CPI-17 to be an active inhibitory form for MP.3. The 5'-flanking region of the human MYPT1 was clone and sequenced. From this analysis MYPT1 gene was found to be a housekeeping gene.4. As MYPT1 has critical functions in MP, we knocked out the gene for MYPT1 in mice to understand the function of MP. In MYPT^<+/-> mice, the levels of MYPT1 expression in smooth muscles were similar to those in wild-type mice. No MFPT1^<-/-> newborns were obtained from the MYPT1^<+/-> intercrosses, indicating that the MYPT1 null mutation was lethal in utero. We will continue to generate the MYPT1-deficient mice using spatio-temporal control system of gene targeting.5. The changes in the expression levels of Rho-kinase-related molecules, namely RhoA, Rho-kinase, CPI-17, myosin light chain kinase, were investigated in various rat hypertensive models compared with normotensive control rats. Although these expression levels were not significantly different, the higher levels of the active form of RhoA were commonly detected in any hypertensive models. These results suggest that regardless of how hypertension begins the activation of RhoA in vascular smooth muscle is one of the principle mechanisms involved is hypertension.

1.从平滑肌中纯化的Rho激酶（ROKα亚型）可磷酸化平滑肌肌球蛋白的调节轻链（Ser 19）和肌球蛋白磷酸酶（MP）的MYPT 1亚基。Rho-激酶可以独立于RhoA被花生四烯酸激活。MYPT 1在Thr 695处的磷酸化诱导MP活性的抑制。使用Thr 695位点特异性和磷酸化特异性抗体，在激动剂刺激的培养的血管平滑肌细胞中观察到Rho激酶在Thr 695处的MYPT 1磷酸化水平增加. CPI-17是MP的抑制性磷蛋白，可被RhoA下游的两种激酶Rho激酶和PKN磷酸化。这些磷酸化将CPI-17转化为MP.3的活性抑制形式。克隆并测序了人MYPT 1的5 '侧翼区。通过分析发现MYPT 1基因是一个管家基因.由于MYPT 1在MP中具有关键功能，我们敲除小鼠中MYPT 1的基因以了解MP的功能。在MYPT^<+/->小鼠中，平滑肌中MYPT 1表达水平与野生型小鼠相似。从MYPT 1 ^<+/->杂交中没有获得MFPT 1 ^<-/->新生儿，表明MYPT 1无效突变在子宫内是致命的。我们将继续利用基因打靶的时空调控系统来产生MYPT 1缺陷小鼠.在各种大鼠高血压模型中与正常血压对照大鼠相比，研究Rho激酶相关分子即RhoA、Rho激酶、CPI-17、肌球蛋白轻链激酶表达水平的变化。虽然这些表达水平没有显著差异，但在任何高血压模型中通常检测到更高水平的RhoA活性形式。这些结果表明，无论高血压如何开始，血管平滑肌中RhoA的激活是高血压的主要机制之一。